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Surface-Enhanced Raman Spectroscopy to Characterize Different Fractions of Extracellular Vesicles from Control and Prostate Cancer Patients
Extracellular vesicles (EVs) are membrane-enclosed structures ranging in size from about 60 to 800 nm that are released by the cells into the extracellular space; they have attracted interest as easily available biomarkers for cancer diagnostics. In this study, EVs from plasma of control and prostate cancer patients were fractionated by differential centrifugation at 5000× g, 12,000× g and 120,000× g. The remaining supernatants were purified by ultrafiltration to produce EV-depleted free-circulating (fc) fractions. Spontaneous Raman and surface-enhanced Raman spectroscopy (SERS) at 785 nm excitation using silver nanoparticles (AgNPs) were employed as label-free techniques to collect fingerprint spectra and identify the fractions that best discriminate between control and cancer patients. SERS spectra from 10 µL droplets showed an enhanced Raman signature of EV-enriched fractions that were much more intense for cancer patients than controls. The Raman spectra of dehydrated pellets of EV-enriched fractions without AgNPs were dominated by spectral contributions of proteins and showed variations in S-S stretch, tryptophan and protein secondary structure bands between control and cancer fractions. We conclude that the AgNPs-mediated SERS effect strongly enhances Raman bands in EV-enriched fractions, and the fractions, EV12 and EV120 provide the best separation of cancer and control patients by Raman and SERS spectra.
Starch and Glycogen Analyses : Methods and Techniques
For complex carbohydrates, such as glycogen and starch, various analytical methods and techniques exist allowing the detailed characterization of these storage carbohydrates. In this article, we give a brief overview of the most frequently used methods, techniques, and results. Furthermore, we give insights in the isolation, purification, and fragmentation of both starch and glycogen. An overview of the different structural levels of the glucans is given and the corresponding analytical techniques are discussed. Moreover, future perspectives of the analytical needs and the challenges of the currently developing scientific questions are included.
Comparison of Multiscale Imaging Methods for Brain Research
A major challenge in neuroscience is how to study structural alterations in the brain. Even small changes in synaptic composition could have severe outcomes for body functions. Many neuropathological diseases are attributable to disorganization of particular synaptic proteins. Yet, to detect and comprehensively describe and evaluate such often rather subtle deviations from the normal physiological status in a detailed and quantitative manner is very challenging. Here, we have compared side-by-side several commercially available light microscopes for their suitability in visualizing synaptic components in larger parts of the brain at low resolution, at extended resolution as well as at super-resolution. Microscopic technologies included stereo, widefield, deconvolution, confocal, and super-resolution set-ups. We also analyzed the impact of adaptive optics, a motorized objective correction collar and CUDA graphics card technology on imaging quality and acquisition speed. Our observations evaluate a basic set of techniques, which allow for multi-color brain imaging from centimeter to nanometer scales. The comparative multi-modal strategy we established can be used as a guide for researchers to select the most appropriate light microscopy method in addressing specific questions in brain research, and we also give insights into recent developments such as optical aberration corrections.
Quantification of EGFR-HER2 Heterodimers in HER2-Overexpressing Breast Cancer Cells Using Liquid-Phase Electron Microscopy
Currently, breast cancer patients are classified uniquely according to the expression level of hormone receptors, and human epidermal growth factor receptor 2 (HER2). This coarse classification is insufficient to capture the phenotypic complexity and heterogeneity of the disease. A methodology was developed for absolute quantification of receptor surface density ρR, and molecular interaction (dimerization), as well as the associated heterogeneities, of HER2 and its family member, the epidermal growth factor receptor (EGFR) in the plasma membrane of HER2 overexpressing breast cancer cells. Quantitative, correlative light microscopy (LM) and liquid-phase electron microscopy (LPEM) were combined with quantum dot (QD) labeling. Single-molecule position data of receptors were obtained from scanning transmission electron microscopy (STEM) images of intact cancer cells. Over 280,000 receptor positions were detected and statistically analyzed. An important finding was the subcellular heterogeneity in heterodimer shares with respect to plasma membrane regions with different dynamic properties. Deriving quantitative information about EGFR and HER2 ρR, as well as their dimer percentages, and the heterogeneities thereof, in single cancer cells, is potentially relevant for early identification of patients with HER2 overexpressing tumors comprising an enhanced share of EGFR dimers, likely increasing the risk for drug resistance, and thus requiring additional targeted therapeutic strategies.
Cold atmospheric plasma is a potent tool to improve chemotherapy in melanoma in vitro and in vivo
Malignant melanoma is a devastating disease. Because of its aggressiveness, it also serves as a model tumor for investigating novel therapeutic avenues. In recent years, scientific evidence has shown that cold atmospheric plasma (CAP) might be a promising modality in cancer therapy. In this study, we aimed to evaluate the effect of CAP generated by an argon plasma jet alone or in combination with dacarbazine (DAC) on melanoma cells in vitro and in vivo. The effects of the CAP on inducing lipid peroxidation and nitric oxide production were higher in B16 melanoma cells in comparison to non-malignant L929 cells. Assays on cell growth, apoptosis, and expression of genes related to, e.g., autophagic processes, showed CAP to have a substantial impact in melanoma cells while there were only minoreffects in L929 cells. In vivo, both CAP monotherapy and combination with DAC significantly decreased tumor growth. These results suggest that CAP not only selectively induces cell death in melanoma but also holds promises in combination with chemotherapy that might lead to improved tumor control. © 2020 by the authors.
Impact of Camera Viewing Angle for Estimating Leaf Parameters of Wheat Plants from 3D Point Clouds
Estimation of plant canopy using low-altitude imagery can help monitor the normal growth status of crops and is highly beneficial for various digital farming applications such as precision crop protection. However, extracting 3D canopy information from raw images requires studying the effect of sensor viewing angle by taking into accounts the limitations of the mobile platform routes inside the field. The main objective of this research was to estimate wheat (Triticum aestivum L.) leaf parameters, including leaf length and width, from the 3D model representation of the plants. For this purpose, experiments with different camera viewing angles were conducted to find the optimum setup of a mono-camera system that would result in the best 3D point clouds. The angle-control analytical study was conducted on a four-row wheat plot with a row spacing of 0.17 m and with two seeding densities and growth stages as factors. Nadir and six oblique view image datasets were acquired from the plot with 88% overlapping and were then reconstructed to point clouds using Structure from Motion (SfM) and Multi-View Stereo (MVS) methods. Point clouds were first categorized into three classes as wheat canopy, soil background, and experimental plot. The wheat canopy class was then used to extract leaf parameters, which were then compared with those values from manual measurements. The comparison between results showed that (i) multiple-view dataset provided the best estimation for leaf length and leaf width, (ii) among the single-view dataset, canopy, and leaf parameters were best modeled with angles vertically at -45⸰_ and horizontally at 0⸰_ (VA -45, HA 0), while (iii) in nadir view, fewer underlying 3D points were obtained with a missing leaf rate of 70%. It was concluded that oblique imagery is a promising approach to effectively estimate wheat canopy 3D representation with SfM-MVS using a single camera platform for crop monitoring. This study contributes to the improvement of the proximal sensing platform for crop health assessment. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.
Eosinophils and Neutrophils-Molecular Differences Revealed by Spontaneous Raman, CARS and Fluorescence Microscopy
Leukocytes are a part of the immune system that plays an important role in the host's defense against viral, bacterial, and fungal infections. Among the human leukocytes, two granulocytes, neutrophils (Ne) and eosinophils (EOS) play an important role in the innate immune system. For that purpose, eosinophils and neutrophils contain specific granules containing protoporphyrin-type proteins such as eosinophil peroxidase (EPO) and myeloperoxidase (MPO), respectively, which contribute directly to their anti-infection activity. Since both proteins are structurally and functionally different, they could potentially be a marker of both cells' types. To prove this hypothesis, UV-Vis absorption spectroscopy and Raman imaging were applied to analyze EPO and MPO and their content in leukocytes isolated from the whole blood. Moreover, leukocytes can contain lipidic structures, called lipid bodies (LBs), which are linked to the regulation of immune responses and are considered to be a marker of cell inflammation. In this work, we showed how to determine the number of LBs in two types of granulocytes, EOS and Ne, using fluorescence and coherent anti-Stokes Raman scattering (CARS) microscopy. Spectroscopic differences of EPO and MPO can be used to identify these cells in blood samples, while the detection of LBs can indicate the cell inflammation process.
On the liquid chemistry of the reactive nitrogen species peroxynitrite and nitrogen dioxide generated by physical plasmas
Cold physical plasmas modulate cellular redox signaling processes, leading to the evolution of a number of clinical applications in recent years. They are a source of small reactive species, including reactive nitrogen species (RNS). Wound healing is a major application and, as its physiology involves RNS signaling, a correlation between clinical effectiveness and the activity of plasma‐derived RNS seems evident. To investigate the type and reactivity of plasma‐derived RNS in aqueous systems, a model with tyrosine as a tracer was utilized. By high‐resolution mass spectrometry, 26 different tyrosine derivatives including the physiologic nitrotyrosine were identified. The product pattern was distinctive in terms of plasma parameters, especially gas phase composition. By scavenger experiments and isotopic labelling, gaseous nitric dioxide radicals and liquid phase peroxynitrite ions were determined as dominant RNS. The presence of water molecules in the active plasma favored the generation of peroxynitrite. A pilot study, identifying RNS driven post‐translational modifications of proteins in healing human wounds after the treatment with cold plasma (kINPen), demonstrated the presence of in vitro determined chemical pathways. The plasma‐driven nitration and nitrosylation of tyrosine allows the conclusion that covalent modification of biomolecules by RNS contributes to the clinically observed impact of cold plasmas. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.
The Effect of Diet and Farm Management on N2O Emissions from Dairy Farms Estimated from Farm Data
The N2O emissions of 21 dairy farms in Germany were evaluated to determine the feasi-bility of an estimation of emissions from farm data and the effects of the farm management, along with possible mitigation strategies. Emissions due to the application of different fertilisers, manure storage and grazing were calculated based on equations from the IPCC (Intergovernmental Panel of Climate Change) and German emission inventory. The dependence of the N2O emissions on fertiliser type and quantity, cultivated crops and diet composition was assessed via correlation analysis and linear regression. The N2O emissions ranged between 0.11 and 0.29 kg CO2eq per kilogram energy-corrected milk, with on average 60% resulting from fertilisation and less than 30% from fertiliser storage and field applications. The total emissions had a high dependence on the diet composition; in particular, on the grass/maize ratio and the protein content of the animal diet, as well as from the manure management. A linear model for the prediction of the N2O emissions based on the diet composition and the fertilisation reached a predictive power of R2 = 0.89. As a possible mitigation strategy, the substitution of slurry for solid manure would reduce N2O emissions by 40%. Feeding cows maize-based diets instead of grass-based diets could reduce them by 14%. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.
Myxobacteria-Derived Outer Membrane Vesicles: Potential Applicability Against Intracellular Infections
In 2019, it was estimated that 2.5 million people die from lower tract respiratory infections annually. One of the main causes of these infections is Staphylococcus aureus, a bacterium that can invade and survive within mammalian cells. S. aureus intracellular infections are difficult to treat because several classes of antibiotics are unable to permeate through the cell wall and reach the pathogen. This condition increases the need for new therapeutic avenues, able to deliver antibiotics efficiently. In this work, we obtained outer membrane vesicles (OMVs) derived from the myxobacteria Cystobacter velatus strain Cbv34 and Cystobacter ferrugineus strain Cbfe23, that are naturally antimicrobial, to target intracellular infections, and investigated how they can affect the viability of epithelial and macrophage cell lines. We evaluated by cytometric bead array whether they induce the expression of proinflammatory cytokines in blood immune cells. Using confocal laser scanning microscopy and flow cytometry, we also investigated their interaction and uptake into mammalian cells. Finally, we studied the effect of OMVs on planktonic and intracellular S. aureus. We found that while Cbv34 OMVs were not cytotoxic to cells at any concentration tested, Cbfe23 OMVs affected the viability of macrophages, leading to a 50% decrease at a concentration of 125,000 OMVs/cell. We observed only little to moderate stimulation of release of TNF-alpha, IL-8, IL-6 and IL-1beta by both OMVs. Cbfe23 OMVs have better interaction with the cells than Cbv34 OMVs, being taken up faster by them, but both seem to remain mostly on the cell surface after 24 h of incubation. This, however, did not impair their bacteriostatic activity against intracellular S. aureus. In this study, we provide an important basis for implementing OMVs in the treatment of intracellular infections.